TRPM7 regulates gastrulation during vertebrate embryogenesis

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca²⁺- and Mg²⁺-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg²⁺ supplementation or by expression of the Mg²⁺ transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg²⁺ in Rac-dependent polarized cell movements during vertebrate gastrulation.     

Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins

The Ca²⁺-binding helix-loop-helix structural motif called “EF-hand” is a common building block of a large family of proteins that function as intracellular Ca²⁺-receptors. These proteins respond specifically to micromolar concentrations of Ca²⁺ in the presence of ~1000-fold excess of the chemically similar divalent cation Mg²⁺. The intracellular free Mg²⁺ concentration is tightly controlled in a narrow range of 0.5-1.0 mM, which at the resting Ca²⁺ levels is sufficient to fully or partially saturate the Ca²⁺-binding sites of many EF-hand proteins. Thus, to convey Ca²⁺ signals, EF-hand proteins must respond differently to Ca²⁺ than to Mg²⁺. In this review the structural aspects of Mg²⁺ binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca²⁺-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg²⁺ ion cannot engage the ligands of an EF-hand in the same way as Ca²⁺ and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg²⁺ plays an active role in the Ca²⁺-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca²⁺ levels. Therefore, some pathological conditions attributed to Mg²⁺ deficiency might be related to excessive activation of underlying Ca²⁺-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.     

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21(Cip1) and p27(Kip1) in renal epithelial NRK-52E cells

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.     

Effect of Noncompetitive Blockade of N-Methyl-d-Aspartate Receptors on the Neurochemical Sequelae of Experimental Brain Injury

Pharmacological inhibition of excitatory neurotransmission attenuates cell death in models of global and focal ischemia and hypoglycemia, and improves neurological outcome after experimental spinal cord injury. The present study examined the effects of the noncompetitive N-methyl-D-aspartate receptor blocker MK-801 on neurochemical sequelae following experimental fluid-percussion brain injury in the rat. Fifteen minutes after fluid-percussion brain injury (2.8 atmospheres), animals received either MK-801 (1 mg/kg, i.v.) or saline. MK-801 treatment significantly attenuated the development of focal brain edema at the site of injury 48 h after brain injury, significantly reduced the increase in tissue sodium, and prevented the localized decline in total tissue magnesium that was observed in injured tissue of saline-treated animals. Using phosphorus nuclear magnetic resonance spectroscopy, we also observed that MK-801 treatment improved brain metabolic status and promoted a significant recovery of intracellular free magnesium concentrations that fell precipitously after brain injury. These results suggest that excitatory amino acid neurotransmitters may be involved in the pathophysiological sequelae of traumatic brain injury and that noncompetitive N-methyl-D-aspartate receptor antagonists may effectively attenuate some of the potentially deleterious neurochemical sequelae of brain injury.     

Nitrogen and potassium fertilization and environmental factors affecting the grass tetany hazard of wheat forage

Summary The effects of temperature and soil moisture levels on the chemical composition of wheat forage grown in growth chambers were studied. In addition to the environmental variables, K and N fertilization effects were studied. In all the studies, increasing levels of K fertilization depressed the Mg and Ca concentration of the shoots. Nitrogen fertilization increased the Mg concentration but had no effect on the Ca concentration of the plants. N fertilization depressed the K concentration in the soil moisture experiment, but had no effect on K concentration in the temperature experiment. Increasing the temperature from 10 to 20°C did not affect the Mg and Ca concentration of the shoots, but the K concentration declined due to dilution effects caused by the greater yield at the higher temperature. In the soil moisture level experiment the K, Mg and Ca concentration in wheat tended to decline with soil moisture level due to dilution effects. Calculations showed that uptake of K was regulated primarily by diffusion of K from the soil to the plant root and that the uptake of Mg was regulated by the uptake process of the plant root and not by the nutrient transport process through the soil.This study was part of the program of the Center for Root-Soil Research. Dept. of Agronomy paper #1532.     

A cytochemical study on the role of ATPases during pollen germination in Agapanthus umbelatus L'Her.

Summary Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca²⁺-ATPases were mainly localized in mitochondria and ER membranes, while Mg²⁺-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K⁺-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.The results are discussed in terms of the ultrastructural organization of the PG and PT, especially those correlated with (1) formation and maintenance of ionic gradients throughout the PT, (2) polarized growth and (3) hydrodynamics of PT elongation.Abbreviations PT Pollen tube - PG pollen grain - PTW pollentube wall - PGW pollen-grain wall - ER endoplasmic reticulum - NEM N-ethylmaleimide